In silico and in vitro prediction of new synthesized N-heterocyclic compounds as anti-SARS-CoV-2

Computer-aided drug design has been employed to get the medicinal effects against Corona virus from different pyridine derivatives after synthesizing the new compounds. Additionally, various computational studies are also employed between the newly prepared pyridine derivatives and three controls against three proteins (6Y2E, 6M71 and 6M3M). Different methods were employed to synthesize new pyridine derivatives according to the literature using different reaction mediums. MTT was performed for cytotoxicity study and IC50 for inhibitory concentration. Additionally, in-silico studies including DFT, molecular docking, molecular dynamics, MMPBSA, ADME, pharmacokinetics and Lipinski rules were evaluated. The chemical structures of all new compounds were elucidated based on spectroscopic investigation. A molecular docking study demonstrated that compounds 5, 11, and 12 have the best binders of the SARS-CoV-2 main protease enzyme, with energy scores of − 7.5 kcal/mol, − 7.2 kcal/mol, and − 7.9 kcal/mol, respectively. The net binding energy values of the 11-Mpro, 12-Mpro, and 5-Mpro complexes revealed their highly stable nature in terms of both intermolecular interactions and docked conformation across the simulation time. ADME properties, besides the pharmacokinetics and Lipinski rules, showed that all seven newly synthesized compounds follow Lipinski rules with high GI absorption. The In Vitro antiviral study against SARS-CoV-2 using MTT methods confirms that compound 5 has more potential and is safer than other tested compounds. The study shows that the newly synthesized pyridine derivatives have medicinal properties against SARS-CoV-2 without violating Lipinski rules. Compounds 5, 11, and 12, particularly compound 5, may serve as promising potential candidate for COVID-19.

calculating the optimized parameters (arrangement of the atoms, chemical bond lengths, angles and optimized structure).The calculated parameters can be used in a variety of experimental and theoretical investigations including vibrations, charge distribution, HOMO and LUMO orbital energies, and many more.Further, druglikeness of the compounds were determined by means of Lipinski's rule of five so that they are suitable drugs to be marketed.

Chemistry
The Key starting active compound, nicotinic acid hydrazide 2, was synthesized according to the literature method 5 .Equimolar reaction of compound 2 with ethyl cyanoacetate gave the corresponding cyanoacetohydrazide derivative 3 35 , which cyclized to the corresponding oxadizole-pyran derivative 5 by refluxing with salicaldehyde in ethanol and catalytic amount of ammonium acetate (Fig. 3).
The Infrared spectrum of compound 5 was devoid of any absorption band in characteristic carbonyl group region, instead, it showed an absorption band at 1652 cm -1 correlated to the C = N group, which confirmed the cyclization step.Furthermore, proof for the assigned structure of 5 was gained from its 1 HNMR spectrum which exhibited only one broad singlet signal for NH group besides multiple signals for aromatic protons.Moreover, its 13 CNMR was in agree with the suggested structure as it exhibited three signals at δ 160.67, 161.21, 163.15 ppm referring to C = N groups (cf.experimental).
On the other hand, synthesis of new pyridine-triazolo-thione derivative 7 was achieved by treating the hydrazide compound 2 with 2-phenylacetyl isothiocyanate at room temperature producing the open adduct thiourea derivative 6, followed by cyclization in refluxing acidic medium (Fig. 3).www.nature.com/scientificreports/ The structure of compounds 6 and 7 were assessed from their spectral data.Their IR spectra represented an additional characteristic band at 1238-1242 cm −1 for C = S group.The newly formed triazole ring in compound 7 was established from its 1 HNMR spectrum which exhibited only one broad absorption band in the downfield region correlated to NH proton.While it represented extra exchangeable broad absorption signals for three NH protons in compound 6. 13 C NMR spectra of compounds 6 and 7 were in agree with the suggested structure (cf.experimental).
New pyridine carbohydrazide compounds 8 and 9 were synthesized by refluxing equimolar amount of compound 2 with cinnamoyl chloride or lauryl chloride in dry benzene (Fig. 4).
The chemical structures of compounds 8 and 9 were confirmed from their spectral data.Their IR spectra showed characteristic absorption bands for C = O and NH, and CH aliphatic in compound 9.Moreover, the predicted structures of compounds 8 and 9 were compatible with their 1 HNMR and 13 CNMR spectra (cf.experimental).
Further formation of pyridine bearing different heterocycles was achieved through formation of thiadiazol derivative 10 from the reaction of nicotinic acid with thiosemicarbazide in acidic medium 36 .
IR spectra of compounds 11 and 12 were devoid of any absorption for amino group, instead, they revealed the presence of bands for C = N groups.Their 1 HNMR displayed one singlet signals corresponding to azomethine proton N = CH as well as one more broad singlet signal in the downfield region correlated to NH proton in case of compound 11.(cf.experimental).

HOMO, LUMO, and chemical reactivity descriptors
The particular kind of binding locations and sites of activity where the protein can be banded have been identified by HOMO and LUMO.The positive and negative nodes are symbolized by a variety of hues (Fig. 7).Here, the blue color in HOMO indicates the positive nodes and yellow color donates negative nodes of MFOs.Secondly, Kelly green is positive and dark red is negative.In case of HOMO and LUMO, the orbital spreading area of LUMO is lower region of HOMO.Pursuant to the FMOs diagram, LUMO has been inhabited by functional groups comprising C-N or N-N bonding, whereas HOMO is slightly found in their region.It is capable of functioning as a  both electrophilic attracting group material as a result of its raised more LUMO in functional groups.The lower magnitude of energy gap contributes to form an interaction with SARS-CoV-2 protein.
The chemical stability of a molecule has determined by the HOMO-LUMO energy gap.The smaller energy gap, the more stable chemical compound with low dynamics 37 and functional group is responsible for this Ref. 38.In the Table 1, the energy gap ranges from − 6.973 to − 7.67 eV with a minimal energy gap.Where energy gap for all compounds is around − 7.0 eV, which represents a lower energy gap as a result, the compounds can be considered as more stable.
Better absorption rate depends on hardness and softness, to provide a better absorption rate the value of hardness should be around − 4.00 eV and softness should be lower than hardness.Here, the highest hardness value is − 3.835 eV with the compound 5 and lower value is − 3.487 eV with 11.This refers that their value is around − 4.00 eV.On the other hand, the value of softness ranges from − 0.261 to − 0.287 eV, which is clearly lower than hardness.According to above data these compounds are stable and have a great absorption rate.Electrostatics potential maps Electrostatics potentiality maps determined the suitable attack side for the compounds whether the compounds will be nucleophilic or electrophilic.The positive and negative sites within the compounds can be determined by this concept 39 .Here, red color demonstrates the positive nodes for 5, 6, 7, 8, 9,11, and 12 and blue color demonstrates the positive nodes for 5. On the other hand, blue color describes negative nodes for all the compounds without 5.Positively charged area is bigger for 5, 7, 9, 12, and then negatively charged area, while negatively charged area is bigger for 6 and 8 than positively charged area (Fig. 8).As a result, the nucleophilic compound is more desirable for those (5, 7, 11, 12, and 9) compounds whereas electrophilic compounds are desirable for 6 and 8.

Molecular docking
Molecular docking is now in routine use application of computer aided drug designing in order to predict intermolecular docking conformation and estimate the binding energy score of docked complexes.The compounds were found to show stable interactions and good affinity for all studies receptors form the SARS-CoV-2 virus.Among the compounds, the 5, 11 and 12 were prioritized as best binders of SARS-CoV-2 main protease enzyme with energy score of -7.5 kcal/mol, -7.2 kcal/mol and -7.9 kcal/mol, respectively (Table 2).All the three compounds were demonstrated to bind the substrate binding pocket of the enzyme with interactions network dominated by both short distance hydrophobic and hydrophilic contacts compared to the studied

Molecular dynamic simulation
The molecular dynamic simulation is a computational based technique to study dynamic properties of biological macromolecule and macromolecule-ligand complexes.The main protease enzyme was seen in considerable stable state in the presence of the compounds as depicted by the root mean square deviation (RMSD) analysis given in Fig. 10A.The mean RMSD of the 11-Mpro complex, 12-Mpro complex and 5-Mpro complex is 1.0 Å, 0.9 Å, and 1.1 Å, respectively.The stable structure of the enzyme illustrates that the compounds binding conformation with the receptor is static and formed multiple strong hydrophilic and hydrophobic contacts.The residue level stability  of receptor molecule was understand using root mean square fluctuation (RMSF), which complements the RMSD and demonstrates overall residues level stable behavior of complexes.The 5-Mpro complex showed some smaller N-terminal jumps that were linked to the loops, which are naturally flexible regions.The average RMSF value of 11-Mpro complex, 12-Mpro complex and 5-Mpro complex is 0.7 Å, 0.8 Å and 1.3 Å, respectively (Fig. 10B).

Binding free estimation
The binding free energies of top ranked complexes were estimated to validate the intermolecular binding interactions and docked stability.The MM-GBSA method is considered more reliable than the conventional docking studies.The Table 3

Evaluation of physicochemical and drug likeness properties (Lipinski Rules)
The most acceptable rules for recognizing drug likeness properties are Lipinski rules which is also known as rules of five.Among the five rules the drug candidate should follow at least four rules to be a drug 40 .Commonly acceptable rules are molecular weight, hydrogen bond acceptors and donors, TPSA and log S with the acceptable values are 150 to 500 g/mol, ≤ 10, ≤ 5, 20 to 130 Å 2 and less than 6, respectively.The molecular weight for all the compounds is in the referenced value.The highest Molecular weight is 351.81 g/mol and the lowest molecular weight is 267.28 g/mol, for the compounds 12 and 8, respectively.The hydrogen bond acceptors range from 3 to 6.All the compounds have lower than 10 hydrogen acceptors..The bioavailability values for the experimented compounds are at 0.55, all the compounds are bioavailable besides that their GI absorption is also high.Table 4 shows that all the experimental compounds are following the drug likeness properties.All of them can be drug candidate in terms of pharmacokinetics and Lipinski rules.

Pharmacokinetics parameters and ADMET properties of pyridine derivatives
ADME is a parameter for a chemical regarding safety as a drug.ADME stands for Absorption, distribution, metabolism, excretion, and Toxicity.The drug candidate should follow the standard of the following: log Kp greater than 0.90 cm/s indicates significant Caco2 permeability.Absorption in the intestines must be greater than 30%, Log Kp greater than − 2.5 indicates minimal skin penetration; for BBB permeability if the log BB value is greater than 0.3, it will readily cross the BBB on the other hand if log BB is less than − 1, it will poorly distributed to the brain, those with a logPS > − 3 are considered to be unable to enter the CNS 42 .
Only 5 and 12 can cross the Caco2 with the same value at 0.993 cm/s, besides these two compounds no compound can cross the Caco2 because their value is less than 0.90 cm/s., 6, 7, 8, 9 and 11 can cross the Caco2 with the value at, 0.867, 0.866, 0.511, 0.511 and 0.858, respectively.Intestinal absorption rat e is more than 30% for all the compounds.Without 6 and 11 the intestinal absorption rate is more than 90%.6 and 11 has 76.423and 88.488% intestinal absorption rate, respectively.5 and 12 has the highest absorption rate at 97.982 and 97.556, www.nature.com/scientificreports/respectively.7, 8 and 9 has the absorption rate at around 90%.The skin permeability value for all the compound is following the standard.All the compounds have the value higher than − 2.6.The log BB value for BBB permeability is lower than − 1 with the value ranges from − 0.014 to − 0.850, the value refers as poorly distributed to the brain, as a result no compound crosses the BBB.Log PS value for CNS permeability ranges from − 2.003 to − 2.759, which considered as the drug cannot penetrate the CNS.No compounds can inhibit CYP2C9, but all the compounds can inhibit the CYP1A2.No compound can excrete Renal OCT2 substrate (Table 5).Toxicity is the harmful effect of drug on living organism 43 .Toxicity of the new synthesized compounds including aquatic and non-aquatic toxicity are listed in Table 6.Only the ligand 7 has the positive AMES toxicity, no other ligands have positive AMES toxicity.5, 6, 7, 11, and 12 have to risk of hepatic impairment because they have negative hepatotoxicity values, but 9 is risky for hepatic impairment.No compounds have skin sensation, they are safe for skin.Maximum tolerated dose for all the compound without 9 is very low.Oral rat acute toxicity, Oral Rat Chronic Toxicity, and T. Pyriformis toxicity are negative for all the compounds.At the end, only one compounds has AMES toxicity (7) and another one compound has Hepatotoxicity (9).Otherwise, rest of the compounds have no adverse effect.5, 6, 11, and 12 have no toxicity, they are safe for the animal health.

In vitro antiviral anti-COVID-19 biological activity
Based on the results obtained from molecular docking and molecular dynamic studies.among the seven tested compounds, the three compounds 5, 11, and 12 with the highest binding energy score were investigated for the term of cytotoxicity concentration 50 (CC50) and Inhibitory concentration 50(IC 50 ) against SARS-CoV-2.
It is well known concept of literature for 50% cytotoxicity concentrations (CC50) carried on the concentration of test compounds necessitated to reduce cell viability by 50%.Accurately, cytotoxicity of the test compounds is unsurpassed governed simultaneously with clean cells to obtain the 50% of CC50 values.However, it can be defined as the concentration of tested sample where 50 percent of the host cells were killed by the antiviral product.We strongly acclaim for determining the 50% of CC50 values in both stationary and dividing cells from multiple germane human cell types and tissues to establish the potential for cell-cycle, species, or tissue-specific toxicities.On the other hand, in-vitro susceptibility of viruses to any types ofantiviral agents is stereotypically and emperically measured as the inhibitory concentration 50% (IC 50 ), So it is may be defined as the concentration for growth for lowers 50% of the virus-induced cytopathic effect (CPE) and the number of plaques formed.

Experimental Chemistry
The instruments used for measuring the melting points, spectral data (IR, mass, 1 HNMR and 13 CNMR) and elemental analysis are provided in detail in the Supplementary Information.Synthesis of nicotinic acid hydrazide derivatives ( 2) and ( 3) were performed according to the reported methods in the literature 5,35

Molecular optimization and evaluation of FMOs, and chemical descriptors
Structure optimization and DFT was permed by material studio version 8.0 with the DMol3 and hybrid functional of B3LYP 45 .
The main rationale of the Becke, 3-parameter Lee-Yang-Parr (B3LYP) method is widely used for DFT calculations because it is highly capable of giving the accurate results for the electronic structure and quantum calculations.The precise estimation of electronic properties, as well as the electron transport potential by evaluating the electrostatic 3D map, was obtained using DFT in this study.The HOMO (highest occupied molecular orbital) and LUMO (lowest unoccupied molecular orbital) are two frontier molecular orbitals that have been used to predict chemical descriptors, such as electrophilicity (ω), chemical potential (μ), electronegativity (Χ), hardness (η), and softness (S).The ionization energy (Ι) and electron affinity (A) can be determined using I = -HOMO and A = -LUMO values, respectively.More details were provided in Supplementary Information.

Molecular docking
The docking study was initiated by retrieving crystal structure of SARS-CoV-2 main protease enzyme (6Y2E), RdRP (6M71) and nucleocapsid protein (6M3M) from protein data bank 46 .The structures were processed in UCSF Chimera v1.16 by subjecting to energy minimization process 47 .Briefly, missing hydrogen atoms were added first and then charge assignment was done using Gasteiger method.The energy minimization was conducted using steepest descent and conjugate gradient methods for total of 1000 steps.On the other side, the compounds were drawn in ChemDraw Ultra 12.0 and then energy minimized using MM2 force field in Chem3D pro 12.0 48 .Molecular docking was performed in PyRx 0.8 using AutoDock 4.0 software 49,50 .The active site information of main protease enzyme, RDRP and nucleocapsid protein was collected from published literature 51,52 .The grid box dimensions set around the active box was set 25 Å along the XYZ axis.The active site residue coordinates used in docking for the main protease enzyme, RdRP and nucleocapsid protein was His41, Asn496 and Phe286, respectively.The number of docked poses generated for each compound was set to 100.The best docked complexes were measured in term of binding energy in kcal/mol.The docked complexes were visualized via Discovery Studio v 2021 53 and UCSF Chimera v 1.16 47 .

Molecular dynamic simulation and MMPBSA analysis
Molecular dynamic simulation was performed for the three top complexes using AMBER20 simulation software 54 .The simulation protocol was accomplished using steps described by Ahmad et al., 2017 55 .The initial processing of the complexes was done through Antechamber program of AMBER20 56 .The receptor protein was prepared using FF14Sb force field while the compounds were prepared via GAFF force field 57 .The molecular dynamics simulations were conducted in three phases; prmtop files generation, preprocessing and production run.The selected docked complexes were first submerged into TIP3P water box, where counter ions were added to get neutral systems.The complexes were then heated gradually to 310 K, then equilibrated and subjected to a production run of 200 ns.The temperature control was achieved using Langevin dynamics 58 .The hydrogen bonds were constrained by SHAKE algorithm 59 .The CPPTRAJ was employed to investigate structure dynamics of complexes 57 .The XMGRACE v5.1 was considered for plotting simulation graphs 60 .The binding free energy of complexes were estimated using MM-GBSA method and accomplished through MMPBSA.pymodule of the AMBER software 61,62 .In total, 1000 frames were picked from the simulation trajectories and analyzed.

Inhibitory concentration 50 (IC 50 ) determination
In 96-well tissue culture plates, 2.4 × 10 4 Vero-E6 cells were distributed in each well and incubated overnight at a humidified 37 °C incubator under 5%CO2 condition.The cell monolayers were then washed once with 1 × PBS and subjected to virus adsorption (hCoV-19/Egypt/NRC-03/2020 (Accession Number on GSAID: EPI_ISL_430820)) for 1 h at room temperature (RT).More details were provided in Supplementary Information.

Ethics declarations
All experiments were performed in accordance with relevant named guidelines and regulations.No human participants/ human cells, tissues or animals were involved in the studies.

Conclusion
Experimental and computational method is used to experiment the medicinal effects of pyridine derivatives against COVID-19.Firstly, Synthesis was performed to get the desired compound by employing different methods.Frontier molecular orbitals and Reactivity descriptor analysis confirms the better stability and absorption rate.Then, molecular docking of seven pyridine derivatives against 6Y2E, 6M71 and 6M3M are performed and get highest docking results at of − 7.5 kcal/mol, − 7.2 kcal/mol and − 7.9 kcal/mol, for 11, 12 and 5, respectively.Furthermore, the molecular dynamics was performed, and it verified the protein-ligand binding is stable.MMGBSA was performed to validate the intermolecular binding interactions and docked stability according to RMSD and RMSF value.This experiment confirms that all the top complexes are highly stable in nature at the time of simulation.It also confirms that 11, 12 and 5 is more stable and active than other.The cytotoxicity and IC 50 test were performed to check the safety and potentiality of the most potent compounds.MTT test confirms compound 5 is safer than other.After that, ADME properties was collected from web server besides that pharmacokinetics and Lipinski rules are also calculated.All the seven compounds follow Lipinski rules, there is no violation and GI absorption is high.To conclude, considering all the factors, compounds 5, 11 and 12 can be potential drug for COVID-19, especially compound 5.

Figure 1 .
Figure 1.Recently reported pyridine and conjugated pyran based heterocyclic compounds with antiviral and anti-coronavirus activities.

Figure 3 .
Figure 3. Synthesis of new pyridine attached oxadiazole and triazole rings.

Figure 5 .
Figure 5. Synthesis of new Schiff base of pyridine-thiadiazol bearing different heterocycles.

Figure 6 .
Figure 6.Optimized structure of the new synthesized compounds.

Figure 8 .
Figure 8. Map of Molecular Electrostatic Potential (MEP) Charge Distribution of reported compounds.

Figure 9 .
Figure 9. Compounds docked conformation with SARS-CoV-2 main protease enzyme.The enzyme is shown by hydrophobic surface, while compounds are in different colors stick.The 11, 12 and 5 is in yellow, pink and blue stick, respectively.The interactions of 11 (a), 12 (b), 5 (c) and control (d) are also given.

Figure 7. (continued)Table 1 .
Frontier molecular orbital of HOMO and LUMO.Frontier molecular orbitals and Reactivity descriptor analysis of the new compounds.

Table 2 .
Docking score of compounds with respect to different SARS-CoV-2 receptors in kcal/mol.
tabulates all the binding energy terms estimated for the complexes.As can be observed, the van der Waals energy dominates the overall net energy of complexes.The van der Waals energy of 11-Mpro Complex, 12-Mpro Complex and 5-Mpro Complex is − 45.12 kcal/mol, − 51.37 kcal/mol and − 47.60 kcal/mol, respectively.Likewise, the electrostatic energy term was also found to favor the intermolecular energy.The electrostatic energy of 11-Mpro Complex, 12-Mpro Complex and 5-Mpro Complex is − 16.10 kcal/mol, − 14.0 kcal/ mol and − 18.54 kcal/mol, respectively.On the other side, the solvation energy in particular the polar solvation energy is less contributing to overall energy.The net solvation energy for complexes is 7.22 kcal/mol (11-Mpro Complex), 8.08 kcal/mol (12-Mpro Complex) and-3.3kcal/mol (5-Mpro Complex).The net binding energy value of the 11-Mpro Complex, 12-Mpro Complex and 5-Mpro Complex is − 54 kcal/mol, − 57.29 kcal/mol and − 62.84 kcal/mol, respectively.These values demonstrate the complexes highly stable nature in term of both intermolecular interactions and docked conformation across the simulation time.

parameter 11-Mpro complex 12-Mpro complex 5-Mpro complex
All the compounds are following the standard TPSA value.8 and 9 have the lowest value at 71.09 Å 2 and 6 has the highest value at 115.21 Å 2 .Log S values are in negative which confirms that all are lower than 6 and following the drug likeness properties.The seven-drug candidate is following all the Lipinski rules, there is no violation of Lipinski rules.0.55 is the standard bioavailability value for a drug Vol:.(1234567890) Scientific Reports | (2024) 14:1152 | https://doi.org/10.1038/s41598-024-51443-7www.nature.com/scientificreports/

Table 4 .
Data of Lipinski rule, pharmacokinetics and drug likeness of different types of pyridine derivatives.

Table 5 .
ADME properties of different types of pyridine derivatives.

Table 6 .
Aquatic and non aquatic toxicity of the new synthesized compounds.